Understanding of the regulation of methionine biosynthesis in Lemna was extended by the following results: a) a number of inhibitors which are specific for steps in various branches of the pathway were developed; b) the previous finding that exogenous methionine inhibits incorporation of sulfate sulfur into methionine was confirmed by an independent method but biological variability has for the moment prevented an unequivocal determination of whether it also inhibits the synthesis of its C4 moiety; c) methionine (or a derivative) non-allosterically regulates the amount and/or activity of cystathionine synthase. Growth in the presence of excess methionine decreases synthase activity 85 percent, whereas growth with limiting methionine increase activity 2- to 4-fold; d) the ratio of cystathionine synthase to O-phosphohomoserine (OPH) sulfhydrylase activity (1:1) remains constant under all growth conditions tested or when inhibited in vitro by proparylglycine. The ratio of O-acetylserine (OAS) sulfhydrylase to OPH sulfhydrylase activities (400:1 in control plants) varies over a 50-fold range under the same growth or inhibitor conditions. These data suggest that OPH sulfhydrylase activity is a property of cystathionine synthase whereas OAS sulfhydrylase is an activity of a separate enzyme.